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K.A. Martemyanov, P.V. Lishko, I.B. Leskov, N. Calero, A.V. Kolesnikov, M.E. Burns, C.J. Chen, V.Y. Arshavsky; R9AP Regulates the Activity of RGS9- Gß5 and Targets its Localization In Vivo . Invest. Ophthalmol. Vis. Sci. 2003;44(13):3178.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: RGS9-Gß5 protein complex regulates the lifetime of activated transducin by stimulating its GTPase activity. The complex is present in the outer segments of photoreceptors where it is tightly bound to the disc membranes via a newly identified protein anchor- R9AP. Our goal was to determine the role of R9AP in (1) regulation of catalytic activity of RGS9-Gß5 and (2) targeting the localization of RGS9-Gß5 to the outer segments. Methods: To study the role of R9AP in determining RGS9-Gß5 localization we created a transgenic mouse where a variant of RGS9-Gß5 with impaired ability to interact with R9AP (RGS9ΔDEP) was expressed on an RGS9 knockout background (RGS9-/-). The localization of R9AP and RGS9 in the rod photoreceptor compartments was studied using the tangential serial sectioning/Western blotting technique. The consequences of RGS9ΔDEP expression on the rod physiology were analyzed using single cell electrophysiological recordings. Regulation of RGS9-Gß5 activity by R9AP was studied using in vitro reconstituted system with purified components. Results: We have shown that recombinant R9AP is able to enhance the ability of RGS9-Gß5 to activate transducin GTPase. In wild type mice both R9AP and RGS9 are localized to the outer segments of rod photoreceptors. In transgenic mice, RGS9ΔDEP was localized predominantly to the inner segment of rods whereas the localization of R9AP was unchanged. Electrophysiological studies of the mouse photoreceptors revealed that the light responses of RGS9ΔDEP transgenic rods were indistinguishable from those of RGS9-/- rods. There were no detectable differences in the time to peak, integration time or final recovery time constant of the dim flash responses, or in the amplitudes of the single photon responses. Conclusions: We conclude that R9AP stimulates the GTPase activating properties of RGS9. The formation of the complex between RGS9-Gß5 and R9AP is essential for the ability of RGS9-1 to localize in the rod outer segments and to efficiently stimulate GTP hydrolysis in the intact rods.
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